Quantifying the Metabolic Activation of Nevirapine in Patients by Integrated Applications of NMR and Mass Spectrometries

Abstract

ABSTRACT:Nevirapine (NVP), an antiretroviral drug, is associated with idiosyncratic hepatotoxicity and skin reactions. Metabolic pathways of haptenation and immunotoxicity mechanisms have been proposed. NVP is metabolized by liver microsomes to a reactive intermediate that binds irreversibly to protein and forms a GSH adduct. However, no reactive metabolite of NVP, trapped as stable thioether conjugates, has hitherto been identified in vivo. This study has defined the metabolism of NVP with respect to reactive intermediate formation in patients and a rat model of NVP-induced skin reactions. An integrated NMR and mass spectrometry approach has been developed to discover and quantify stable urinary metabolite biomarkers indicative of NVP bioactivation in patients. Two isomeric NVP mercapturates were identified in the urine of HIV-positive patients undergoing standard antiretroviral chemotherapy. The same conjugates were found in rat bile and urine. The mercapturates were isolated from rat bile and characterized definitively by NMR as thioethers substituted at the C-3 and exocyclic C-12 positions of the methylpyrido ring of NVP. It is proposed that NVP undergoes bioactivation to arene oxide and quinone methide intermediates. The purified major mercapturate was quantified by NMR and used to calibrate a mass spectrometric assay of the corresponding metabolite in patient urine. This is the first evidence for metabolic activation of NVP in humans, and only the second minimum estimate in patients of bioactivation of a widely prescribed drug associated with idiosyncratic toxicities. The method can be used as a template for comparative estimations of bioactivation of any drug in patients.

Evidence for nevirapine bioactivation in man: Searching for the first step in the mechanism of nevirapine toxicity

“…Although NVP mercapturates have previously been identified by Srivastava et al (2010) in the urine of HIV-positive individuals, it should be noted that these species may not mirror accurately the full extent of NVP bioactivation. Indeed, HIV-infected individuals have depleted GSH levels (Townsend et al, 2003).…”

“…Besides providing clues about the molecular mechanisms underlying drug toxicity, the covalent adducts formed upon reaction with bionucleophiles can be useful markers of toxicity for biomonitoring purposes, especially when well-characterized standards are available (Törnqvist et al, 2002;Angerer et al, 2007;Rubino et al, 2009). Srivastava et al (2010) have provided evidence for covalent binding of reactive NVP metabolites to amino acids and peptides (presumably GSH) in vivo, by identifying two NVP mercapturate conjugates in the urine of HIV-positive individuals. However, evidence for NVP metabolism to reactive derivatives with haptenation ability in humans, yielding stable protein adducts, has yet to be provided.…”

The importance of hapten–protein complex formation in the development of drug allergy

“…These reactive intermediates are electrophilic and have the potential to modify nucleophilic residues in proteins. Identification of two isomeric nevirapine mercapturates in the urine of patients and rats has provided the evidence that nevirapine undergoes bioactivation to arene oxide and quinone methide intermediates in vivo [30][31][32].…”

Quantitative estimation of circulating metabolites without synthetic standards by ultra‐high‐performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction

“…[1,2] The major limitation for this approach is that it relies on the availability of radiolabeled compounds. Ultraviolet (UV) [3,4] and nuclear magnetic resonance (NMR) [4][5][6] have been used to calibrate MS response of metabolites relative to parent compounds using biologically generated metabolite standards. Although less sensitive, both detectors have a more universal response than MS. NMR has been found to be more accurate than UV, but it requires the isolation of a few micrograms of metabolites, which can be challenging in the case of minor metabolites from complex biological matrices.…”